Proteins are present in many commercially available preparation forms. In particular, proteins are present as the active ingredient in some pharmaceutical medicaments. For the preparation of these preparation forms, it is beneficial to use the proteins in crystalline form. As well as being easier to handle in crystalline form proteins as dry crystals are, more stable than, for example, proteins in dissolved form.
As an example, the preparation of a pharmaceutical preparation containing the hormone insulin as active ingredient, uses the protein insulin in crystalline form. Crystallized insulin is stable, for example, at a temperature of xe2x88x9220xc2x0 C. over a number of years.
Crystalline forms of proteins having a molecular weight of up to several hundred thousand daltons and peptides having a lower molecular weight are known. The amino acid sequence of the proteins can either be identical to a naturally occurring sequence or it can be changed relative to the natural form. In addition to containing amino acid chains, the proteins can also contain sugar radicals or other ligands as side chains. The proteins may be isolated from natural sources, or the protein may be prepared by genetic engineering or synthetically, or the proteins may be obtained by a combination of these processes.
In aqueous solution, proteins have a three-dimensional structure of greater or lesser complexity which is based on a specific spatial folding of the amino acid chains. The intact structure of a protein is essential for its biological action. During crystallization of the proteins from aqueous solutions, this structure is largely retained. The crystal structure of a protein is predominantly determined by its amino acid sequence. Another factor determining crystal structure is, for example, inclusions of low molecular weight substances, such as, for example, metal ion salts and water molecules. In particular, the presence of a certain amount of intracrystalline water molecules (water of crystallization) is necessary for the stability of the crystal structure of proteins.
For example, insulin crystals require an optimum residual water content (roughly between 1 and 7%). If the crystals are overdried and too low a moisture content is achieved, then the water of crystallization has already been removed from the crystals. As a result, the chemical stability of insulin is adversely affected, resulting, for example, in the formation of higher molecular weight compounds. It is assumed that the higher molecular weight fractions in the insulin are responsible for immunological incompatibility reactions. In the extreme case, insulin can be denatured to the extent that the crystals are no longer soluble in aqueous media. If, on the other hand, crystals with too high a moisture content are obtained upon drying, then there is too much water between the individual crystals. The insulin is partly dissolved in this intercrystalline water. The stability of the dissolved insulin is, however, significantly lower than that of solid forms.
It is known that protein crystals, in particular insulin crystals, can be dried by isolating the crystals from a crystal suspension by filtration and drying the filter cake under reduced pressure at a temperature above 0xc2x0 C. It is also known that the drying process can be accelerated by replacing the intercrystalline water with ethanol prior to drying. During drying, the crystals are distributed in a thin layer on drying sheets or are agitated on the filter.
In another process, the protein crystals, for example insulin crystals, are frozen as an aqueous suspension at a temperature below 0xc2x0 C. on drying sheets and then freeze-dried under reduced pressure.
In both processes mentioned, the attainment of neither a defined nor optimal residual moisture content in the dried crystals is adequately ensured. In both processes, the drying process is kinetically controlled to the end and must be terminated at a specific time in order to avoid overdrying. Experience shows that it is difficult to determine the correct point in time to terminate the drying. The resulting moisture content of the crystals depends not only on the thickness of the layer on the drying sheet, but also on the size of the crystals (or their surface area). Since the crystallization process leads to insulin crystals having a size distribution of varying width, the crystals dried in a kinetically controlled process consist of a mixture of dryer small crystals and moister large crystals.
Another disadvantage of these processes is that it is very difficult to automate charging and emptying of the equipment used for drying. Thus, the operation of charging and emptying still largely requires manual work, which harbors the risk of contamination by germs and foreign particles. For example, the currently valid pharmacopeia demand that crystalline insulin for the preparation of pharmaceutical preparations must be low in germs but not germ-free. Therefore a need exists for a process of preparing a protein preparation where the process provides crystals with a uniform water content without the risk of contamination.
The present invention comprises a process for drying protein crystals starting from an aqueous protein crystal suspension, which comprises drying the protein crystal suspension in a centrifugal dryer.
In one embodiment of the invention the process for drying protein crystals starting from an aqueous protein crystal suspension encompasses filtering off the protein crystals from the aqueous protein crystal suspension, and subsequently bringing the protein crystals into a drying medium which consists of a mixture of water and a nonaqueous solvent which is miscible with water in any ratio and which has a lower vapor pressure than water.
The present invention involves a process for preparing a protein preparation which preferably comprises the following steps:
1. Filtering off the crystals from an aqueous suspension.
2. Washing the filter cake.
3. Replacing the wash liquid with a drying medium.
4. Spindrying the filter cake.
5. Detaching the filter cake from the filter and converting it into a fluidized bed.
6. Drying the crystals in the fluidized bed with a stream of moistened nitrogen.
7. Emptying the dried crystals using a nitrogen pressure surge into a flanged container.
Accordingly, the present invention relates to a process for drying protein crystals starting from an aqueous protein crystal suspension, which comprises drying the protein crystal suspension in a centrifugal dryer.
For the drying process described, it is particularly advantageous if the detached filter cake can be converted into a fluidized bed. The experiments, carried out in a commercially available dryer, have shown that if the intercrystalline water was not replaced for one of the drying media described below, it was not possible to generate a fluidized bed; a mixture of crystal aggregates of varying size formed instead, making uniform drying impossible.
Other experiments have shown that by replacing the intercrystalline water with a pure nonaqueous drying medium, e.g. 100% strength ethanol or 100% strength propanol, only the intracrystalline water (water of crystallization) remaining in the protein crystals, is it possible to produce a fluidized bed. However, during the subsequent drying phase, the drying medium could not adequately be removed even after a prolonged drying time. The dried products had a residual content of drying medium of more than 5 %. On the other hand, some of the water of crystallization had already been removed during the drying time.
Surprisingly, it has now been found that by replacing the intercrystalline water with a drying medium which consisted of a mixture of water and a nonaqueous substance, it was possible to avoid the disadvantages mentioned. After replacing the intercrystalline water with one of the drying media described below, it was possible to produce a fluidized bed from the detached filter cake and to dry the crystals satisfactorily. Using moistened drying gas, after a drying time of from about 1 to 4 hours, products were obtained which were free from crystal aggregates and have a residual content of the nonaqueous substances of less than 0. 1%.
Accordingly, the present invention relates to a process for drying protein crystals starting from an aqueous protein crystal suspension, wherein, in particular, the protein crystals, which have been filtered off from the protein crystal suspension, are brought into a drying medium which consists of a mixture of water and a nonaqueous solvent which is miscible with water in any ratio and which has a lower vapor pressure than water. The process according to the invention for drying a protein crystal suspension is advantageously carried out in a centrifugal dryer.
The nonaqueous substances which constitutes part of the suitable drying media have a low vapor pressure at a temperature of about 40xc2x0 C. (i.e. have a lower vapor pressure than water), are miscible with water in any ratio and are chemically inactive toward proteins under specified conditions. Alcohols, such as, for example, methanol, ethanol, n-propanol and isopropanol, are preferable for this use. Mixtures of alcohols are also suitable.
The proportion of nonaqueous substances in the suitable mixtures with water in the drying medium is from about 10% to about 80% inclusive, preferably from 15% to about 60% inclusive, further preferably from about 20% to about 80% inclusive.
In the drying process described using dry nitrogen as drying gas, the drying process is kinetically controlled to the end, meaning that the disadvantages described above can occur.
Our own experiments using moist nitrogen as the drying gas have shown that the removal of the water from the crystals during the drying phase comes to a standstill and the achieved residual water content in the dried crystals is determined by the water content in the drying gas, i.e. at the end of the drying phase a thermodynamic equilibrium between the water content in the drying gas and the water content in the crystals is established. The residual water content in the dried crystals is therefore independent of their size (or surface area).
In addition, the experiments using dry nitrogen as drying gas have shown that the nonaqueous constituent of the drying medium could not be sufficiently removed, even after a prolonged drying period. The dried products had a residual content of the nonaqueous constituent of more than 1 %.
Surprisingly, it has been found that in a process for drying protein crystals using a drying gas, the residual content of the nonaqueous constituent of the drying medium could be removed except for less than 0.1% if a water containing drying gas is used. The drying process is preferably carried out in a centrifugal dryer. In the process for drying protein crystals, it is preferable if the protein crystals, after having been filtered off from the protein crystal suspension, are brought into a drying medium which consists of a mixture of water and a nonaqueous solvent which is miscible with water in any ratio and which has a lower vapor pressure than water. It is more preferable in the process if the protein crystals, after they have been filtered off from the protein crystal suspension, are converted into a fluidized bed for the purpose of drying.
The inventors""experiments with the drying process described have shown that if a sterile crystal suspension free from foreign particles was used, and wash water, drying medium and drying gas were sterilized by filtration, and the centrifugal dryer was suitably cleaned and sterilized, a dried product was obtained which was both germ-free and free from foreign particles.
The process of the present invention is particularly suitable for drying crystals of animal insulin, human insulin or an analog thereof.
Insulin analogs referred to here are preferably derivatives of naturally occurring insulins, namely human insulin or animal insulin, which differ from the corresponding otherwise identical naturally occurring insulin by virtue of substitution of at least one naturally occurring amino acid radical and/or addition of at least one amino acid radical and/or organic radical.